GENE VARIANTS & THEIR INTERACTIONS DEFINING HUMAN NTD RISK Neural tube defects (NTDs), primarily spina bifida and anencephaly, arise from a complex interplay of multiple gene interactions and environmental exposures. After 30 years of clinical and basic research, the field remains unable to accurately predict the risk for an individual couple of having a child affected by NTD, how folic acid (FA) works to prevent NTDs, whether or what dose of FA is likely provide effective prevention for them or whether there is another nutrient/supplement or intervention that would provide greater benefit The recent confluence of information from genetic mouse models, capabilities of molecular biological and biochemical detection in embryonic systems and advances in genomics and computational genetics now provides sufficient power to successfully address this complex genetic disorder. Project 1 will test the following hypotheses: 1. that combinations of rare variant single nucleotide polymorphisms (SNPs) will display associations useful for the definition of individual NTD risk in humans, and 2. that recognition of interactions between these genetic patterns with environmental conditions, including FA intake and factors common to inflammation or oxidative/nitrosative stress, can further increase their predictive value. This project will use deep resequencing of NTD patient DNA, targeted to human counterparts of some 1,000 genes implicated in NTD pathogenesis by clinical and animal model studies, to identify rare variant alleles that are overrepresented in NTD patients. These will be used to design custom SNP assays for screening larger patient numbers for analyses of single gene and pair-wise associations with NTD. Computational modeling will assess the potential impact of NTD associated SNPs on key developmental and metabolic pathways. The functional significance of SNP associations in humans will be functionally tested first for impact on Wnt/PCP, FA metabolism and oxidative/nitrosative stress using in vitro and mouse systems assays that will also be used to validate and inform computational modeling. Because the overt NTD phenotypes are readily recognized in humans and experimental animals, NTDs may well be the first complex genetic disorder for which gene-gene and gene-environment interactions can be understood in depth. Progress made for this disorder can provide useful analytical tools for identifying molecular network interactions relevant to later-onset complex genetic disorders, like schizophrenia and autism. RELEVANCE (See instructions): Enhanced capabilities for assessment of individual risk for developing NTDs would permit prevention regimens to be tailored to individuals rather than applied 'shot-gun' to populations. In broadest scope, data generated in Project 1 will have implications for every reproductive-age woman worldwide. In addition, folate metabolism can exert a lasting impact on gene expression by influencing DNA methylation, making it imperative that we understand the ramifications of FA supplementation. A fuller grasp of the relationships between the FA pathway and risk genes will have important relevance for a broad range of other diseases in which folate status may have a role.